Despite of a great deal of effort by public and private institutions, we don't have a prophylactic vaccine against HIV-1 or an eradication protocol to treat infected patients. Recently, laboratories all over the word have reported an increase in the transmission of HIV-1 strains that are resistant to one or more components of the antiretroviral (ART) cocktail. In order to find novel therapeutic targets, basic understanding of HIV-1 replication is urgently needed. Early steps in the replication of human immunodeficiency virus (HIV-1) involve delivery of the viral core into the cytoplasm of the host cell. The viral core is a conical structure composed of ~1500 monomers of the HIV-1 capsid protein, which houses the viral RNA genome. We studied HIV-1 uncoating, reverse transcription, and nuclear import by exploiting the ability of different cellular factors to affect early events of HIV-1 replication. ?Uncoating? is the dissociation of monomeric capsids from the HIV-1 core. Proteins and drugs that destabilize the HIV-1 core and accelerate uncoating in the cytosol prevent reverse transcription. Consistent with the concept that reverse transcription is linked to uncoating, we have shown that genetic or pharmacologic inhibition of reverse transcription prevents uncoating. During the previous funding period, we found that cellular proteins that stabilize the HIV-1 core allow reverse transcription but not nuclear import. These results support a model in which the HIV-1 core is a ?container? that holds the elements required for reverse transcription. Opening the ?container? disrupts reverse transcription. Keeping the ?container? closed allows completion of reverse transcription but prevents nuclear import. These results suggest that core stability is tightly modulated during infection. Cyclophilin A (CypA) accounts for up to 0.4% of cellular protein and interacts with the HIV-1 capsid and core. However, the role that CypA plays in HIV-1 infection is not known. Our preliminary data indicate that CypA depletion inhibits HIV-1 infection in primary CD4+ T cells by 100 fold, supporting the use of this model to understand the role of CypA in HIV-1 infection. We hypothesize that CypA functions as a central modulator of HIV-1 infection by controlling interactions between cellular factors and the HIV-1 core during the early steps of HIV-1 infection. With the expert support of Dr. Hultquist, Dr. Campbell, Dr. Luban and Dr. KewalRamani, the following specific aims will test this hypothesis. Aim 1 will determine the mechanisms through which CypA modulates HIV-1 infection in human primary CD4+ T cells. Aim 2 will determine the effects of CypA on the HIV-1 capsid in the nucleus. Aim 3 will examine the roles of other cyclophilins in HIV-1 replication. Aim 4 will determine the effects of the Nup358 cyclophilin domain (Cyp358) on HIV-1 nuclear import. This proposal will address two important questions on the HIV-1 field: 1) The role of CypA in HIV-1 replication, which has been elusive for 25 years, and 2) the role of nuclear capsid in HIV-1 productive infection.